1. Field of the Invention
The present invention relates to the field of medical arts; more particularly to methods of determining cancer cell sensitivity to acylfulvene class alkylating agents and to insights for design and identification of anticancer alkylating prodrugs.
2. Background
Cancer is second only to heart disease as a leading cause of death in the United States. Cancer occurs when cells continue to divide and fail to die at the appropriate time. Under normal circumstances, the many types of cells that make up the body grow and divide to produce more cells as they are needed in order to maintain a healthy state. Despite notable success in cancer treatments, current therapies have not yet significantly affected mortality rates for some of the more common cancers. The most common therapies include radiation and drug treatments, often used in combination. At present there are only a relatively small number of clinically active anti-cancer compounds, many of which have limited activity and/or are toxic to healthy cells.
Alkylating agents are a class of such drugs that trigger cell death by covalently binding to cellular nucleophiles such as DNA and proteins. Several alkylating agents, such as cyclophosphamide, ifosphamide and mytomycin C, are prodrugs that require metabolic activation to unmask inherent electrophilicity of an effective functional group. Selective toxicity to cancer cells is due to specific uptake of the activated metabolite (e.g., cyclophosphamide and ifosphamide) or by activation by an enzyme or by various substances in the cellular environment.
Alkylating agents exert their action by interacting with DNA, damaging the DNA so that it can no longer replicate, generally by DNA strand breakage or intra- or interstrand crosslinking of bases. Some agents may act by adding alkyl groups to DNA bases, formation of cross bridges between DNA molecules and inducing mutations arising from mispairing; however, agents causing mispairing are considered undesirable because this may lead to increased malignant proliferation (cancer). Types of alkylating agents include mustards, ethylenimes, alkylsulfonates, triazines, piperazines and nitrosureas. Cyclosporamide is used in cancer therapies and is thought to be selectively activated to some extent by cancer cells, so that they are relatively more toxic to cancer cells than to normal cells.
Illudins are natural products, first isolated from the toxic Jack O'Lantern mushroom. They were identified as candidates for new cancer agents (Kelner, et al., 1990) and used to further identify a sub-class of less toxic chemotherapeutics, the acylfulvenes (Kelner, et al. Cancer Chemotherapy and Pharmacology, 1999) of which 6-hydroxymethylacylfulvene (HMAF) was identified as a lead compound. Despite improved effectiveness over the illudins, it was recognized that little was known how the acylfulvenes work in the body and that identification of other more effective acylfulvene-related compounds was an important goal.
Many alkylating compounds have been tested for anticancer activity; however, most are unable to preferentially kill cancer cells. As noted, irofulven is a member of the acylfulvene class of compounds, which has performed well in tests of HT29 and MV522 induced tumors in mice (MacDonald, et al., 1997; Britten, et al., 1999. Although some clinical trials have been initiated, the toxicity of irofulven continues to be a major concern.
Irofulven is a semisynthetic derivative of the mycotoxin illudin S. The sesquiterpenes illudin S and illudin M are unique toxins produced by the bioluminescent Jack O'lantern mushroom Omphalotus illudens and related species. They are potent antibacterial and antitumor compounds. Illudin S exhibits cytotoxic and cytostatic properties at nanomolar concentrations in several human tumor cell lines in vitro (Kelner, et al., 1997). It is actively transported into cells where it is thought to form protein and DNA adducts (Kelner, et al., 1990). Adduct formation in turn leads to inhibition of DNA synthesis, single-stranded DNA breaks, cell cycle arrest, and onset of apoptosis.
Human tumor cells with various multiple drug resistance phenotypes are known to be as sensitive to illudin S as parental non-multiple drug resistant lines, thus indicating promise as anticancer agents. Unfortunately, substantial systemic toxicity was shown in animal models, raising serious concerns for use in cancer treatments (Kelner, et al., 1987). In a search for related compounds with greater therapeutic indices, irofulven was synthesized, proving to be nearly 2 orders of magnitude less cytotoxic than illudin S. While clearly a more promising candidate for cancer chemotherapy than illudin S, irofulven has similar drawbacks because of significant toxicity, including therapeutic levels that are close or overlap with toxicity levels. Many patients therefore cannot be treated with this drug due to myelosuppression and renal dysfunction.
HMAF is believed to act as an alkylating agent by forming DNA, RNA, and protein adducts that are preferentially cytotoxic to human cancer cells. Despite the conspicuous differences in cytotoxicity between illudin S and irofulven, it has long been assumed that the same molecular mechanism causes their cytotoxic effect (McMorris, et al., 1996). Studies of illudin and acylfulvene metabolites isolated from reactions with rat liver cytosol (Tanaka, et al., 1990; McMorris, et al., 1999) led to the suggestion that reduction of the carbon-carbon double bond of the α,β-unsaturated ketone of an illudin or acylfulvene would lead to an extremely unstable electrophilic cyclohexadiene intermediate. Attack of the cyclopropyl group by a cellular nucleophile would then lead to adduct formation, dysfunction of the adducted macromolecule, and ultimately cell death. Although chlorinated and hydroxylated metabolites were isolated and thought to represent reaction of the putative electrophilic intermediate with chloride anion and water nucleophiles, respectively (Tanaka, et al., 1990), no adduct was directly observed.
The improved efficacy of HMAF over the parental illudins has led to attempts to develop second and third generation compounds that are less toxic, yet maintain cancer cell selectivity. Numerous acylfulvenes and illudin analogs have been prepared, including those disclosed in U.S. Pat. No. 6,025,328; U.S. Pat. No. 5,932,553; U.S. Pat. No. 6,548,679; each of which is herein incorporated by reference in its entirety. It is not known which, if any, of these analogs is less cytotoxic to normal cells than the parent irofulven.
Deficiencies in the Art
Despite the wide use of chemotherapy, there are several major drawbacks due to the chemical nature of some of these drugs. Ideally, cancer treatments will cure the cancer, but most chemotherapeutics control the disease for a limited amount of time before losing effectiveness. Exactly why this happens is not known; however, chemotherapy remains one of the most used and often effective methods of cancer treatment. An important tool in developing cancer drugs is an understanding of the mechanism of drug action against a cancer cell. This will be a key in identifying and developing chemotherapeutics that are highly toxic toward the cancer but relatively non-toxic toward non-cancerous cells.
Selection of lead compounds for testing as chemotherapeutic drug candidates is often a time-consuming and inexact process. Rapid screening tests to evaluate old and new generation drugs are needed; this is particularly important for compounds that have promise as therapeutics but are unacceptably toxic to normal, healthy cells. In particular, rapid in vitro tests are needed to evaluate potential efficacy of acylfulvenes that are being developed as second and third generation irofulven alkylating agents for cancer chemotherapy.